Process for recovering amino acids from protein hydrolysates

ABSTRACT

Amino acid fractions of different composition are obtained at different times by fractional filtration after neutralization of the hydrolysates. Tyrosine-rich and leucine-rich fractions are specifically treated and lead to the recovery of L-tyrosine, L-cystine, L-leucine and L-phenyl-alanine.

This is a division of application Ser. No. 327,593, filed Dec. 4, 1981,now U.S. Pat. No. 4,384,136 which in turn is a continuation ofapplication Ser. No. 119,111, filed Feb. 6, 1980, now abandoned.

This invention relates to a process for recovering amino acids fromprotein hydrolysates.

For recovering amino acids from vegetable or animal proteins, theproteins first have to be split up into their constituents, i.e. intothe individual amino acids. In the known acid hydrolysis process, theproteins are subjected to prolonged heating with hydrochloric acid orsulphuric acid. Other known processes include hydrolysis of the proteinswith alkalis and enzymatic hydrolysis.

Various specific precipitation methods have been proposed with a view torecovering amino acids from protein hydrolysates. Aromatic sulphonicacid, picric acid, mercury and copper salts are used for theseprecipitations. These processes are seriously complicated by the need torecover the precipitation reagents and by the use of organic solvents.

Separation of the amino acids by distillation of the ethyl esters israrely used nowadays. Analytical separation of the amino acids can becarried out very effectively with ion exchangers. In the preparativerecovery of amino acids, however, difficulties are caused by the largeseparation columns required, by the heavy dilution of the solution andby the contamination of the amino acid solutions by the buffersubstances used for elution.

The present invention is the outcome of research to find a newseparation process for recovering amino acids from protein hydrolysateswhich is simpler and which gives a greater yield at less cost, i.e. maybe worked on an industrial scale.

The process according to the invention is characterised in that, afterneutralisation of the hydrolysates, amino acid fractions of differentcomposition are obtained at different times by fractional filtration.

It has been found that, during the neutralisation of a proteinhydrolysate, the sparingly soluble amino acids crystallise out atdifferent rates. The first fraction which precipitates immediately afterneutralisation is enriched with leucine whilst the later fractionsobtained after cyrstallisation times of at least 3 days are enrichedwith tyrosine and cystine. The separation of the sparingly soluble aminoacids into at least two fractions is a feature of the present invention.

The starting material used for carrying out the process according to theinvention may be obtained from a variety of protein sources rich intyrosine, leucine, isoleucine and phenyl-alanine, such as for exampledesugared molasses, cereal or maizegerm, oil cake micro-organisms,particularly yeasts, or casein. It is preferably obtained by the acidhydrolysis of protein sources such as these, optionally after theremoval of ballast substances.

Preferred further stages of the separation process according to theinvention are described in the following:

Separation of cystine and tyrosine

The separation of cystine and tyrosine presents difficulties because, intheir dipolar form, both these amino acids are very sparingly soluble inwater. It has been found that, in changing from the dipolar to the anionform, the solubility of cystine increases more quickly than that oftyrosine in a certain pH-range. Thus, at a pH-value of 7.0, 0.04% oftyrosine and 0.02% of cystine are in solution, whereas at a pH-value of9.2 0.04% of tyrosine and 1.0% of cystine are in solution. By virtue ofthese changes in the solubility of the amino acids tyrosine and cystine,in a relatively narrow pH-range of 9.0 to 10.0, it is possible toseparate these two amino acids.

Thus, one possible method of recovering L-tyrosine is characterised inthat a fraction rich in tyrosine is dissolved at a pH-value of from 10to 11, the resulting solution is filtered and the tyrosine isprecipitated at a pH-value of from 8.5 to 10, preferably 9.5, separatedoff and purified by recyrstallisation.

One possible method of recovering L-cystine is characterised in that thesolution obtained after separation of the tyrosine is adjusted to apH-value of from 3.0 to 6.0 and the cystine precipitated is separatedoff and purified by recrystallisation.

Separation of leucine, isoleucine, methionine and tyrosine

The two isomeric amino acids, leucine and isoleucine, have very similarphysical properties and are therefore difficult to separate.

It has been found that, in changing from the dipolar to the cation form,the solubility of isoleucine increases more quickly than that of leucinein a certain pH-range. In a saturated sodium chloride solution, 0.4% ofleucine and 0.5% of isoleucine are in solution at a pH-value of 6.0. Ata pH-value of 1.5, 1.1% of leucine and 1.7% of isoleucine are insolution. By adjusting the solution to a specific isoleucineconcentration, leucine can be precipitated at pH 1.0 to 2.0. Additionaldifficulties arise where methionine is present because these amino acidsform co-precipitates with leucine. Accordingly, the methionine isconverted into soluble methionine sulphoxide by oxidation, preferablybefore precipitation of the leucine.

Thus, one possible method of recovering L-leucine is characterised inthat a fraction rich in leucine is dissolved in acid, the methionine isoxidised by the addition of oxidising agents and a crude leucine isprecipitated at a pH-value of from 1.0 to 2.0, the isoleucine content ofthe solution preferably being adjusted to between 1.0 and 1.5%.

The exact pH-value in the range from 1.0 to 2.0 may be selected independence upon the tyrosine content of the leucine-rich fraction. Theleucine may be further purified by repeating the precipitation processin the same pH-range.

Extraction of isoleucine, phenyl-alanine, tyrosine and leucine

The mother liquors from the precipitation of leucine containing theamino acids isoleucine, phenyl-alanine, tyrosine and residues ofleucine. It has been found that the hydrochlorides of these amino acidsmay be extracted in very good yields from solutions containingsodiumchloride using a water-immiscible alcohol, particularly isobutanolor butanol. The extraction of amino acids and amino acid hydrochloridesfrom aqueous solutions using butanol or isobutanol is known. However,this extraction gives poor yields because the distribution coefficientsof the amino acids or amino acid hydrochlorides in the butanol-watersystem are below 1.0. These distribution coefficients may be increasedto a value above 10 by saturating the solution with sodium chloride.

Thus, one possible method of extracting leucine, isoleucine,phenyl-alanine and tyrosine is characterised in that a solution of thehydrochlorides of these amino acids saturated with sodium chloride isextracted with a water-immiscible alcohol, particularly isobutanol orbutanol.

The valine and tyrosine content of the alcohol extract may be furtherreduced by washing the extract with water. This washing with water maybe carried out directly at the head of the extraction column or in aseparate apparatus. Following the addition of water, the alcohol may beazeotropically distilled off from the alcohol extract and returned tothe extraction column. The hydrochloric acid may be removed from theaqueous solution of the amino acid hydrochlorides using a weakly basicanion exchanger and a mixture of leucine, isoleucine, phenyl-alanine andtyrosine may be obtained after concentration of the neutral solution.

Recovery of phenyl-alanine

Normally, phenyl-alanine is recovered together with tyrosine byadsorption on active carbon from acid solution. This process is attendedby various disadvantages. Thus, the active carbon has to be activated bytreatment with acetic acid, the quantity of phenyl-alanine adsorbed,based on the volume of active carbon, is very small and thephenyl-alanine has to be eluted with organic solvents, such as pyridineor aniline.

Accordingly, it is preferred in accordance with the invention to adsorbphenyl-alanine and tyrosine by treating a neutral salt-free amino acidsolution with a strongly basic anion exchanger in hydroxide form,because it has been found that adsorption in this way enables five timesthe quantity of phenyl-alanine per unit volume to be taken up bycomparison with adsorption on carbon.

Thus, one possible method of separating phenyl-alanine and tyrosine ischaracterised in that a neutral solution containing the hydrochloridesof these amino acids is passed over a weakly basic and over a stronglybasic anion exchanger.

It has also been found that it is possible, simply by eluting thestrongly basic anion exchanger with a hydrochloric acid solution,completely to remove the phenyl-alanine and the tyrosine from the ionexchanger. Where regeneration is carried out in the usual way using a 4%sodium hydroxide solution, the phenyl-alanine and tyrosine are onlypartly removed.

The eluates of the anion exchanger contain the hydrochlorides of thephenyl-alanine and tyrosine. Verification of the solubility behaviour in6 n HCl showed that phenyl-alanine hydrochloride is sparingly soluble inthe cold and highly soluble under heat. Tyrosine is more readily solublein 6 n HCl in the cold. Thus, the eluate may be concentrated, itshydrochloric acid content adjusted to between 20 and 25%, sodiumchloride separated off at 70° to 90° C. and the phenyl-alaninehydrochloride crystallised out by cooling. The phenyl-alaninehydrochloride may then be purified by recrystallisation and thehydrochloric acid removed by means of a weakly basic anion exchanger.Finally, pure L-phenyl-alanine may be obtained by concentrating theneutral solution by evaporation.

EXAMPLE 1

An acid protein hydrolysate is neutralised and the leucine-rich aminoacid mixture which crystallises out is immediately filtered off. After acrystallisation time of at least 3 days, a second fraction of thesparingly soluble amino acids is separated off. This amino acid mixtureis dissolved in dilute sodium hydroxide at pH 10.0 and the cystineconcentration is adjusted to between 1.0 and 1.5% by the addition ofwater. After filtration of the solution, the tyrosine is precipitated ata pH-value of 9.5. After a crystallisation time of 24 hours, the crudetyrosine is filtered off, washed with water, dissolved in acid andprecipitated under heat at pH 1.5 to 1.8. Under these precipitationconditions, a crystalline pure tyrosine is obtained.

The filtrate of the crude tyrosine is adjusted with acid to a pH-valueof from 7.0 to 2.0 and preferably to a pH-value of 5.0 and, after 24hours, the crude cystine which has crystallised out is separated off.This crude cystine is dissolved in acid and residues of tyrosine areadsorbed by the addition of active carbon. After filtration, the cystineis precipitated at a pH-value of 2.0 and separated off.

EXAMPLE 2

After neutralisation of a protein hydrolysate, the sparingly solubleamino acids precipitated are separated off as a first fraction rich inleucine. This amino acid mixture is dissolved at a pH-value of 0.5 andthe isoleucine concentration is adjusted to approximately 1.5% by theaddition of water. After the methionine content has been determined, themethionine is oxidised at 90° C. by the addition of an equimolarquantity of hydrogen peroxide. The solution is then filtered after theaddition of active carbon, the pH-value is adjusted with dilute sodiumhydroxide to between 1.0 and 1.5 and, after cooling, the crude leucinewhich has crystallised out is separated off. The crude leucine isre-dissolved at pH 0.5 and further purified by precipitation at pH 1.0to 2.0. This process is repeated until the required purity of theL-leucine is reached.

The choice of the pH-value is determined by the tyrosine content of thesolution. For tyrosine contents of the solution of 0.65; 0.8; 1.0; 1.2;1.4; 1.6 and 1.8%, pH-values of 1.0; 1.65; 1.52; 1.42; 1.33; 1.26 and1.16, respectively, are selected.

EXAMPLE 3

The filtrate from the precipitation of leucine is adjusted withhydrochloric acid to a pH-value of 0.5 and optionally saturated withsodium chloride. This solution is extracted in countercurrent withisobutanol in an extraction column. The ratio of isobutanol to the aminoacid solution is adjusted to 1:1 or 1:2. In this process, the aminoacids leucine, isoleucine and phenyl-alanine are extracted to a level ofapproximately 90%. The amino acids valine and tyrosine are extracted toa level of approximately 50%. The valine and tyrosine content is furtherreduced by washing the isobutanol extract with water. This washing withwater is directly carried out at the head of the extraction column.After the addition of water, the isobutanol is azeotropically distilledoff from the isobutanol extract and returned to the extraction column.The hydrochloric acid is removed from the aqueous solution of the aminoacid hydrochlorides using a weakly basic anion exchanger and a mixtureof leucine, isoleucine and phenyl-alanine is obtained afterconcentration of the neutral solution.

EXAMPLE 4

A mixture of the amino acid hydrochlorides of leucine, isoleucine,phenyl-alanine and tyrosine obtained by extraction with isobutanol ispassed over a weakly basic anion exchanger in order to separate thehydrochloric acid. The neutral amino acid mixture is passed over astrongly basic anion exchanger in the hydroxide form. A pure solution ofleucine and isoleucine is obtained in the liquid issuing from thecolumn. Pure isoleucine may be obtained from this solution in knownmanner by extracting the copper complexes with methyl alcohol.

EXAMPLE 5

The anion exchanger column charged with phenyl-alanine and tyrosine isregenerated with one bed volume of 10% hydrochloric acid and then withone bed volume of 4% sodium hydroxide solution. The eluates from thehydrochloric acid regeneration are collected and concentrated. The acidconcentration is adjusted to at least 20% by the addition ofconcentrated hydrochloric acid and the sodium chloride present isseparated off at a temperature of from 70° to 90° C. After cooling ofthe solution, phenyl-alanine hydrochloride crystallises out and ispurified by recrystallisation from 6 n HCl. To recover freephenyl-alanine, the phenyl-alanine hydrochloride is dissolved in waterand the hydrochloric acid is removed by means of a weakly basic anionexchanger. Pure L-phenyl-alanine crystallises out on concentration ofthe neutral solution.

We claim:
 1. A process for recovering leucine, isoleucine,phenyl-alanine, tyrosine and valine from an acid protein hydrolysatecontaining amino acids comprising the steps of:(a) forming a solution ofamino acid hydrochlorides from the acid protein hydrolysate; (b)saturating the solution with sodium chloride; and then (c) extractingfrom the saturated solution the hydrochlorides of leucine, isoleucine,phenyl-alanine, tyrosine and valine with a water-immiscible alcohol. 2.A process for separating phenyl-alanine and tyrosine from a solution ofamino acid hydrochlorides containing phenyl-alanine hydrochloride andtyrosine hydrochloride comprising the steps of:(a) passing the solutionof amino acid hydrochlorides over a weakly basic anion exchanger toobtain a neutral solution of amino acids; and then (b) passing thesolution of amino acids over a strongly basic anion exchanger ofhydroxide form thereby adsorbing and separating the phenyl-alaninne andtyrosine from the solution of amino acids.
 3. A process for recoveringamino acids from an acid protein hydrolysate comprising the steps of:(a)forming a solution of amino acid hydrochlorides from the acid proteinhydrolysate; (b) saturating the solution with sodium chloride; (c)extracting from the saturated solution the hydrochlorides of leucine,isoleucine, phenyl-alanine, tyrosine and valine with a water-immisciblealcohol; (d) removing the alcohol solvent from the extract by azeotropicdistillation with water to form an aqueous solution of the extractedhydrochlorides; (e) passing the aqueous solution of extractedhydrochlorides over a weakly basic anion exchanger to obtain an aqueoussolution of leucine, isoleucine, phenyl-alanine, tyrosine and valine;(f) passing the aqueous solution of leucine, isoleucine, phenyl-alanine,tyrosine and valine over a strongly basic anion exchanger in hydroxideform thereby adsorbing the phenyl-alanine and tyrosine on said exchangerand obtaining an eluate of an aqueous solution containing leucine,isoleucine and valine; (g) eluting the adsorbed phenyl-alanine andtyrosine from the strongly basic anion exchanger with dilutehydrochloric acid; (h) concentrating the eluate of step (g); (i)adjusting the hydrochloric acid concentration of the concentrated eluateto between 20 and 25%; and then (j) heating the eluate to a temperatureof 70° to 90° C. to separate sodium chloride, and thereafter cooling thehot eluate to crystallize phenyl-alanine hydrochloride.
 4. The processof claims 1 or 3, wherein the alcohol is selected from the groupconsisting of butanol and isobutanol.
 5. The process of claims 1 or 3,wherein the tyrosine and valine content of the alcohol extract isreduced by washing the extract with water.
 6. The process of claim 3,wherein the phenyl-alanine hydrochloride is purified byrecrystallisation and L-phenylalanine is obtained by treatment with aweakly basic anion exchanger.